Failure of appropriate placental development, particularly in the first trimester, is associated with significant complications of pregnancy, including miscarriage, pre-eclampsia and intrauterine growth restriction (IUGR). During the first trimester, placental trophoblast cells undergo dynamic cellular events, including cell proliferation, differentiation, migration/invasion, and cell-cell interactions. These events are required for the formation of the anchoring villous and the invasion of extravillous trophoblast (EVT) deep into the uterine wall where they induce a remodelling of the maternal spiral arteries. In other tissues, intercellular communication via gap junctions is known to modulate these processes, though there is no information as to whether this occurs in the placenta. Therefore, the overall objective of this project is to define the role of gap junctional intercellular communication (GJIC) in placental development. We hypothesize that a loss of GJIC is necessary for EVT to exit the proliferative phenotype of the anchoring villous and invade into the uterine wall. Furthermore, we hypothesize that re-expression of GJIC by populations of invasive EVT induces their terminal differentiation allowing them to associate with endothelial cells in spiral arteries during remodelling, and also, in EVT distant from vessels, to promote their aggregation and fusion to form large, multi-nucleated non-invasive trophoblast. Support for these hypotheses is provided by our preliminary data, using first trimester placental explants, in which we have shown that blockade of GJIC or antisense knock-down of the gap junction protein, connexin40, induces EVTs to differentiate from the proliferative to the invasive phenotype. We will address these hypotheses through 4 specific aims. In Aim I, we will use the explants to determine whether GJIC-blockade alone is sufficient to induce the invasive EVT phenotype and to identify putative upstream modifiers and downstream effectors of GJIC-mediated EVT differentiation. Complementary studies in Aim II will take advantage of the power of trophoblast stem cells to define connexins that modulate differentiation to invasive trophoblast, determine whether mutation of these connexins disrupts normal differentiation and use micro-array analysis to identify trophoblast genes whose expression is modified by GJIC. In Aim Ill, we will utilize a trophoblast-decidua explant co-culture system to determine how the decidua modifies GJICmediated EVT differentiation. In Aim IV, we will use primary trophoblasts and the Jeg-3 cell line (wild type or following stable transfection with connexin proteins) to investigate, in vitro and in vivo, the requirement for GJIC during EVT-endothelial coupling and vessel invasion. This novel investigation integrates the experience and efforts of three laboratories, each recognized internationally for their independent investigations on placental development.